羊膜移植治疗兔早期角膜碱烧伤IGAM-1的表达

目的:探讨新鲜羊膜移植术在早期角膜碱烧伤治疗中抑制炎症促进角膜上皮愈合的作用机理,并与保存羊膜作比较。方法:以36只新西兰大白兔制作角膜碱烧伤模型,并分为新鲜羊膜移植组、保存羊膜移植组和对照组。术后7,14d应用计算机图像分析系统对角膜表达细胞粘附分子-1穴ICAM-1)进行半定量测定。结果:新鲜羊膜移植组角膜炎症反应最轻,白细胞浸润最少熏角膜表达ICAM-1明显低于保存羊膜组。结论:羊膜移植可通过降低角膜组织ICAM-1的表达水平来抑制炎症反应,且这一作用新鲜羊膜要明显优于保存羊膜。     

Intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1 (LFA-1) expression in human epiretinal membranes

Various histogenetically different cell types such as macrophages, retinal pigment epithelial cells, glial cells, and fibroblasts are involved in the formation of epiretinal membranes. In the development of such multicellular tissues, cellular adhesion molecules (CAMS) are necessary for cell migration, proliferation, and localization, and the transfer of information between the cells. We investigated the expression of the intercellular adhesion molecule 1 (ICAM-1) and the leukocyte function-associated antigen 1 (LFA-1) in frozen sections of epiretinal membranes in proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR), macular pucker, and recurrent membranes after intraocular silicone oil tamponade using the indirect immunoperoxidase method. ICAM-1 forms a receptor-ligand pair with LFA-1 and is involved in a number of significant cellular interactions, e.g. in providing dynamic position-specific information to guide lymphocyte and leukocyte localization in the immune response. ICAM-1 is a member of the immunoglobulin gene superfamily of CAMs. LFA-1 is a member of the integrin family of cell membrane receptors. It mediates a wide range of lymphocyte, monocyte, natural killer cell, and granulocyte interactions with other cells in immunity and inflammation, and it is a receptor for ICAM-1. The LFA-1 interaction with its ligand ICAM-1 mediates not only cell adhesion but also signal transduction in immunologic and inflammatory cell responses. Basal ICAM-1 expression is normally low on nonhematopoietic cells, but it can be subject to an up-and-down regulation by various cytokines. ICAM-1 expression was found in 8/9 PVR membranes, in 9/10 PDR membranes, in 4/4 macular pucker, and in 7/8 recurrent membranes after intraocular silicone oil tamponade. Coexpression of LFA-1 was detected in all but one of the ICAM-1-positive membranes. The high frequency of ICAM-1 /LFA-1 coexpression in epiretinal membranes indicates an important role in the pathogenesis of epiretinal membrane formation.Supported by the Herman-Wacker-Fonds, Foundation for Research in Retinal Detachment, Federal Republic of GermanyCorrespondence to: H.-P. Heidenkummer     

In vivo corneal confocal microscopy in keratoconus

PURPOSE: To evaluate the corneas of keratoconic subjects using in vivo confocal microscopy. METHODS: Slit scanning confocal microscopy was used to evaluate the central cornea of one eye of each of 29 keratoconic subjects (mean age 31 +/- 10 years; range 16-49 years). Quantitative aspects of corneal morphology were compared against data from control subjects. RESULTS: Compared with normal control corneas, epithelial wing cell nuclei were larger (p < 0.0001) and epithelial basal cell diameter was larger (p < 0.05) in the keratoconic cornea. Many of the keratoconic corneas investigated showed increased levels of stromal haze and reflectivity, which appeared to be related to the presence of apical scarring on slit lamp examination. A grading scale was devised to quantify the levels of haze. This scale was shown to provide a measure of the level of scarring present. The anterior keratocyte density (AKD) and posterior keratocyte density were 19% lower (p < 0.0001) and 10% lower (p = 0.004) than in controls, respectively. The reduction in AKD was significantly associated with three factors: a history of atopy, eye rubbing and the presence of corneal staining. The mean endothelial cell density in keratoconus was 6% greater than that of normal controls (p = 0.05). The level of endothelial polymegethism was shown not to be different between keratoconic subjects and matched controls (paired t-test: t = 1.82, p = 0.08). CONCLUSIONS: Confocal microscopy demonstrates significant quantitative alterations of corneal morphology in keratoconus.     

活体共聚焦显微镜在角膜病临床研究中的新进展

活体共聚焦显微镜能在细胞水平实时、非侵入性、高清晰地检测角膜结构,它已广泛应用于角膜病的研究.本文对活体共聚焦显微镜在感染性角膜炎、圆锥角膜、角膜后沉着物、长期应用抗青光眼药物引起的角膜病变及糖尿病相关的角膜病变的临床研究新进展进行综述.     

In vivo fluorescence microscopy of corneal neovascularization

· Purpose: The purpose of our study was to establish an animal model to study the microcirculation in corneal neovascularization in the living animal atraumatically. · Methods: Corneal neovascularization was induced in New Zealand white rabbits by a standard micropocket assay utilizing pellets with 250 ng basic fibroblast growth factor. Anesthesia consisted of intramuscular injections of ketamine and xylazine. Intravital microscopy was performed without preparation of the cornea. Rhodamine 6G was used as fluorescent dye to stain leukocytes. Fluorescein-isothiocyanate-dextran served as plasma marker. Microcirculation analysis was done off-line by digital video imaging with special analysis software and included the following parameters: vessel diameters, blood velocity, and differentiation of leukocytes according to their interaction with endothelium into free-floating, rolling and sticking leukocytes. · Results: Vessel diameters in venular trunk vessels showed diameters of 54.0 ± 13.3 μm with 1.1 ± 0.5 mm/s flow; 29.4 ± 16.3% of all leukocytes were attached to the vascular wall. The number of sticking leukocytes was found to be 17.8 ± 36.0 cells/mm² endothelial surface. Values are given for arteriolar trunk and branch as well as venular branch vessels. · Conclusions: This method for in vivo microscopic observation and quantification of the vasculature of the ocular surface seems to be suitable for evaluation of microhemodynamic and leukocyte measurements in mature neovascular vessels. It allows atraumatic experiments without corneal preparation procedures which disturb the microcirculation. The results concerning microhemodynamics and adherence of leukocytes are in a range comparable to other microcirculation studies. This new model could provide insight into the pathophysiology of microcirculatory disorders of the anterior eye segment, e.g. during angiogenesis. Received: 5 June 1997 Revised version received: 22 July 1997 Accepted: 5 August 1997     

In vivo corneal confocal microscopy in marfan syndrome

PURPOSE: To examine the cornea of patients with Marfan syndrome in comparison with a control group by using the in vivo confocal microscope. METHODS: Twenty-four eyes of 12 patients with Marfan syndrome had their corneas examined using the in vivo confocal microscope Heidelberg Retina Tomograph (HRT) II/Rostock Cornea Module. The control group included 24 eyes of 12 subjects who had their corneas examined by the same in vivo confocal microscope. RESULTS: Epithelium and neural plexus examination did not show any difference between the 2 groups. Examination of the stroma showed no significant differences concerning the morphology and density of keratocytes. The extracellular matrix of 16 of the 24 eyes of the Marfan group was clearly visible and showed thin highly reflective interconnected lines between keratocytes. In the healthy eye group, reflective lines were observed in only 5 of the 24 eyes. The endothelium of 14 corneas of the Marfan group showed brightly reflective particles. In no cornea of the control group were such particles observed. CONCLUSIONS: Highly reflective extracellular matrix of the stroma and brightly reflective particles among the endothelial cells were the 2 main corneal findings observed by using in vivo corneal confocal microscopy in patients with Marfan syndrome compared with a control group. Further studies need to be made to confirm these findings and eventually find new criteria for Marfan syndrome from the examination of in vivo corneal confocal microscopy.     

角膜移植排斥反应中ICAM-1含量的变化

目的:探讨细胞间粘附分子-1(intercellular adhesion mole-cule-1,ICAM-1)在血液和房水中的含量变化与角膜移植排斥反应的关系。方法:缝线法诱发兔角膜新生血管化(corneal neovascular-ization,CNV)后,制作穿透性角膜移植(penetrating kerato-plasty,PKP)模型4组,A组:正常对照组;B组:自体穿透性角膜移植组;C组:同种异体穿透性角膜移植组;D组:新生血管化穿透性角膜移植组。术后记录植片存活时间和移植排斥指数(rejection index,RI);ELISA法检测房水及外周血中可溶性ICAM-1(sICAM-1)的含量,免疫组化法观察ICAM-1的表达。结果:B、C组在观察期内未见排斥反应发生。D组发生排斥反应,角膜植片平均存活12.4±1.3d,术后房水及外周血中sICAM-1含量即升高,至排斥反应前达最高水平分别为53.9±19.3ng/L,378.8±30.6ng/L,在排斥反应发生时,免疫组化染色发现角膜组织中ICAM-1呈强阳性表达。结论:血液和房水中的ICAM-1含量可以是角膜移植免疫排斥反应发生的指标,术后监测ICAM-1浓度变化对排斥反应的发生有一定预测和早期诊断作用。     

In vivo confocal corneal microscopy after keratoplasty

BACKGROUND: Seven eyes with clear grafts after penetrating keratoplasty were examined with in vivo confocal corneal microscopy in 1999. Our aim was the confocal microscopic investigation of the subclinical changes in clear grafts after long-term follow-up.METHODS: The preoperative diagnoses were keratoconus (two), granular corneal dystrophy (two), pseudophakic bullous keratopathy due to ACL (two), and corneal ulcer (one). The epithelium, corneal nerves, keratocytes of the anterior and posterior stroma, and endothelium were evaluated with confocal microscopy.RESULTS: Mean density of basal epithelial cells was 3928+/-378 cells/mm(2) at 15 months and 3284+/-565 cells/mm(2) at 66 months postoperatively. At 15 months the keratocyte density was 750+/-113 cells/mm(2) in the anterior stroma and 601+/-98 cells/mm(2) in the posterior stroma, at 66 months 383+/-53 cells/mm(2) in the anterior stroma and 411+/-98 cells/mm(2) in the posterior stroma. Endothelial cell density decreased from 1719+/-576 cells/mm(2) (15 months) to 965+/-272 cells/mm(2) (66 months).CONCLUSIONS: In the follow-up period a significant decrease of keratocyte and endothelial cell density was detectable with confocal microscopy. The clinical importance of our findings must be clarified with further examinations on more patients.     

In vivo assessment of mechanisms controlling corneal hydration

The endothelial pump and evaporation components of corneal recovery were studied in the in vivo human cornea by inducing corneal swelling with the use of hypoxia and monitoring the subsequent decrease in corneal thickness. Corneal recovery follows a nonlinear time course with the rate of recovery decreasing as the cornea thins. Following 60 micron of induced edema, recovery with the eyes open required an average of 2.5 hr to reach baseline corneal thickness, while recovery with the eyes closed took an average of 4.0 hr to reach the normal physiologic corneal swelling (17 micron). Our analysis indicates that for open eye recovery from 60 micron of swelling, the endothelial pump provides 20%, while the osmotic thinning caused by tear evaporation contributes 80% of recovery. During recovery, the rate of water evaporation from the anterior corneal surface remained relatively steady at 2.5 microliter/cm2 X hr. Comparison of measured vs calculated recovery rates during recovery with the eyes closed suggests that the endothelial pump functions at one speed and that the "pump-leak" theory of corneal hydration control is applicable for the human cornea.     

ICAM-1含量变化与兔角膜移植排斥反应的实验研究

目的：观察细胞间粘附分子-1（intercellular adhesionmolecule-1,ICAM-1）含量变化与角膜移植排斥反应的关系。 方法：利用缝线诱发新西兰白兔角膜新生血管（corneal neovascularization,CNV）后,制作穿透性角膜移植（penetrating keratoplasty,PKP）模型4组,术后记录移植排斥指数（rejection index,RI）;记录植片存活时间;应用酶联免疫吸附（ELISA）双抗体夹心法检测房水及外周血中sICAM-1的含量,免疫组织化学染色法观察ICAM-1的表达。 结果：排斥组植片平均存活12.4±1.3d;正常房水和血清中均可检测到少量的sICAM-1,它们的浓度分别为（16.6±3.6）ng/L（,95.2±6.3）ng/L。术后排斥组房水及外周血中sICAM-1含量即升高,至排斥反应前达最高水平分别为（53.9±19.2）ng/L,（378.8±30.6）ng/L,在观察期内维持高水平,排斥反应发生时,免疫组化染色ICAM-1呈强阳性表达。 结论：ICAM-1在角膜移植免疫排斥反应中发挥重要作用,术后外周血中sICAM-1浓度变化对排斥反应的发生有一定预测和早期诊断作用。     

VEGF可溶性受体sFlt-1在角膜新生血管中的作用研究进展

血管内皮生长因子(VEGF)可溶性受体(sFlt-1)是具有抗血管化作用的强力因子之一.新近发现其在正常角膜中亦有表达,并以高亲和中和抗体的形式与内源性VEGF-A结合,阻断其信号传导,抑制其促血管化作用.sFlt-1由Flt-1蛋白的胞外前6个结构域加上1个特异性31氨基酸尾部组成,是Flt-1的mRNA经交替拼接生成,且由于不存在跨膜区,sFlt-1以游离形式存在.在新生血管化角膜中,其含量及存在形式均发生改变,但其始动因素仍不明确.就近年有关sFlt-1的来源、存在形式、在角膜新生血管发生发展中的作用进展及可能的应用前景进行综述.     

Endothelial leukocyte adhesion molecule-1 in endotoxin-induced uveitis.

Expression of endothelial leukocyte adhesion molecule-1 (ELAM-1) on endothelial cells leads to the attachment of polymorphonuclear leukocytes. The sequential expression of ELAM-1 and major histocompatibility complex (MHC) class II antigen was examined in the eyes of 59 Lewis rats with endotoxin-induced uveitis (EIU) after the injection of Salmonella typhimurium endotoxin. The eyes were enucleated at 2-hr intervals. Hematoxylin and eosin-stained paraffin-embedded sections and immunohistochemically stained cryostat sections were graded by two masked observers. The MHC class II antigen was expressed on cells in the iris and ciliary body 4 hr after injection of endotoxin and on the corneal endothelium, 8 hr postinjection. It was found that ELAM-1 was expressed first on cells of the ciliary body and iris 10 hr after the injection of endotoxin and on the corneal endothelium, 22 hr postinjection. Clinical and histopathologic disease developed 16 hr postinjection. Adherence of polymorphonuclear cells to the corneal endothelium was observed at the time of ELAM-1 expression. In conclusion, expression of ELAM-1 on ocular tissue occurred in EIU and appeared to promote polymorphonuclear cell accumulation in the anterior segment of the eye.     

In vivo corneal and conjunctival epithelial nuclear stain

Epithelium plays a very important structural and functional role in the cornea and conjunctiva. Evaluation of the epithelium is the first step in diagnosing many pathologic states. We have developed a new technique for the in vivo staining of nuclei of corneal and conjunctival epithelium in rabbits and guinea pigs. Several drops of 0.01%, 0.1%, 0.25%, 0.5%, or 1.0% toluidine blue or 1.0% methylene blue were applied to the conjunctival sac to stain epithelial cells. The cells picked up the vital dye within 5 minutes and could be photographed at 30X with the Keeler-Konan wide-field specular microscope. Cells and nuclei were clearly observable. Photographs could be further enlarged to enhance details. Wash out time was rapid and no toxic effects were observed. This technique adds a new dimension to the study of epithelium in normal and pathologic states in experimental animals. This technique may also be applicable to human eyes for discerning such diseases as carcinoma, herpes simplex, or superior limbic keratoconjunctivitis.     

人类白细胞抗原DR分子和细胞间粘附分子-1在正常和病变角膜的表达及意义

目的　探讨正常及病变角膜中人类白细胞抗原ＤＲ 分子和细胞间粘附分子１ 的表达及其在角膜炎症和移植排斥反应中的作用和影响。方法　应用免疫组化技术对１０ 例正常人及４８ 例患者的病变角膜标本进行人类白细胞抗原ＤＲ分子和细胞间粘附分子１ 的表达的检测。结果　正常人角膜无或轻微表达人类白细胞抗原ＤＲ 分子和细胞间粘附分子１ ;病变角膜,尤其是炎症角膜及角膜移植术后发生排斥反应的角膜,其阳性表达率明显增高。结论　人类白细胞抗原ＤＲ 分子和细胞间粘附分子１ 的异常表达与角膜炎性反应有关,并可促进移植排斥反应的发生